Postnatal development of mouse spermatogonial stem cells as determined by immunophenotype, regenerative capacity, and long-term culture-initiating ability: a model for practical applications.

Spermatogonial stem cells (SSCs) are the foundation of life-long spermatogenesis. While SSC research has advanced greatly over the past two decades, characterization of SSCs during postnatal development has not been well documented. Using the mouse as a model, in this study, we defined the immunophenotypic profiles of testis cells during the course of postnatal development using multi-parameter flow cytometry with up to five cell-surface antigens. We found that the profiles progress over time in a manner specific to developmental stages. We then isolated multiple cell fractions at different developmental stages using fluorescent-activated cell sorting (FACS) and identified specific cell populations with prominent capacities to regenerate spermatogenesis upon transplantation and to initiate long-term SSC culture. The data indicated that the cell fraction with the highest level of regeneration capacity exhibited the most prominent potential to initiate SSC culture, regardless of age. Interestingly, refinement of cell fractionation using GFRA1 and KIT did not lead to further enrichment of regenerative and culture-initiating stem cells, suggesting that when a high degree of SSC enrichment is achieved, standard markers of SSC self-renewal or commitment may lose their effectiveness to distinguish cells at the stem cell state from committed progenitors. This study provides a significant information resource for future studies and practical applications of mammalian SSCs.

Dissecting the spermatogonial stem cell niche using spatial transcriptomics.

Spermatogonial stem cells (SSCs) in the testis support the lifelong production of sperm. SSCs reside within specialized microenvironments called "niches," which are essential for SSC self-renewal and differentiation. However, our understanding of the molecular and cellular interactions between SSCs and niches remains incomplete. Here, we combine spatial transcriptomics, computational analyses, and functional assays to systematically dissect the molecular, cellular, and spatial composition of SSC niches. This allows us to spatially map the ligand-receptor (LR) interaction landscape in both mouse and human testes. Our data demonstrate that pleiotrophin regulates mouse SSC functions through syndecan receptors. We also identify ephrin-A1 as a potential niche factor that influences human SSC functions. Furthermore, we show that the spatial re-distribution of inflammation-related LR interactions underlies diabetes-induced testicular injury. Together, our study demonstrates a systems approach to dissect the complex organization of the stem cell microenvironment in health and disease.

Spermatogonial Transplantation.

Spermatogonial transplantation is the unequivocal method to detect spermatogonial stem cells (SSCs) based strictly on the functional definition of stem cells - the cells' regenerative capacity. This method further allows for SSC quantification. A weakness of spermatogonial transplantation is its time-consuming nature; it takes 2 months to confirm the production of terminally differentiated cells in spermatogenesis, spermatozoa, in mice, which gives the assay endpoint. Using the mouse as the model system, we here describe the basic techniques of spermatogonial transplantation and provide practical guidance to successfully carry out this technique and to interpret data generated.

Screening of Potential Plasticizer Alternatives for Their Toxic Effects on Male Germline Stem Cells.

Plasticizers give flexibility to a wide range of consumer and medical plastic products. Among them, phthalate esters are recognized as endocrine disruptors that target male reproductive functions. With this notion, past studies designed and produced alternative plasticizers that could replace phthalates with limited toxicity to the environment and to male reproductive functions. Here, we focused on one reproductive cell type that was not investigated in past studies-spermatogonial stem cells (SSCs)-and examined in vitro the effects on 22 compounds (seven plasticizers currently in use and 15 newly synthesized potential alternative plasticizers) for their effects on SSCs. Our in vitro compound screening analyses showed that a majority of the compounds examined had a limited level of toxicity to SSCs. Yet, some commercial plasticizers and their derivatives, such as DEHP (di-(2-ethylhexyl) phthalate) and MEHP (mono-(2-ethylhexyl) phthalate), were detrimental at 10-5 to 10-4 M. Among new compounds, some of maleate- and fumarate-derivatives showed toxic effects. In contrast, no detrimental effects were detected with two new compounds, BDDB (1,4 butanediol dibenzoate) and DOS (dioctyl succinate). Furthermore, SSCs that were exposed to BDDB and DOS in vitro successfully established spermatogenic colonies in testes of recipient mice after transplantation. These results demonstrate that SSC culture acts as an effective platform for toxicological tests on SSC function and provide novel information that two new compounds, BDDB and DOS, are alternative plasticizers that do not have significant negative impacts on SSC integrity.

Constitutive activation of CTNNB1 results in a loss of spermatogonial stem cell activity in mice.

Spermatogenesis requires that a careful balance be maintained between the self-renewal of spermatogonial stem cells (SSCs) and their commitment to the developmental pathway through which they will differentiate into spermatozoa. Recently, a series of studies employing various in vivo and in vitro models have suggested a role of the wingless-related MMTV integration site gene family/beta-catenin (WNT/CTNNB1) pathway in determining the fate of SSCs. However, conflicting data have suggested that CTNNB1 signaling may either promote SSC self-renewal or differentiation. Here, we studied the effects of sustained CTNNB1 signaling in SSCs using the Ctnnb1tm1Mmt/+; Ddx4-CreTr/+ (ΔCtnnb1) mouse model, in which a stabilized form of CTNNB1 is expressed in all germ cells. ΔCtnnb1 mice were found to have reduced testis weights and partial germ cell loss by 4 months of age. Germ cell transplantation assays showed a 49% reduction in total functional SSC numbers in 8 month-old transgenic mice. In vitro, Thy1-positive undifferentiated spermatogonia from ΔCtnnb1 mice formed 57% fewer clusters, which was associated with decreased cell proliferation. A reduction in mRNA levels of genes associated with SSC maintenance (Bcl6b, Gfra1, Plzf) and increased levels for markers associated with progenitor and differentiating spermatogonia (Kit, Rarg, Sohlh1) were detected in these cluster cells. Furthermore, RNAseq performed on these clusters revealed a network of more than 900 genes regulated by CTNNB1, indicating that CTNNB1 is an important regulator of spermatogonial fate. Together, our data support the notion that CTNNB1 signaling promotes the transition of SSCs to undifferentiated progenitor spermatogonia at the expense of their self-renewal.

Yes-associated protein expression in germ cells is dispensable for spermatogenesis in mice.

Yes-associated protein (YAP), a key effector of the Hippo signaling pathway, is expressed in the nucleus of spermatogonia in mice, suggesting a potential role in spermatogenesis. Here, we report the generation of a conditional knockout mouse model (Yapflox/flox ; Ddx4cre/+ ) that specifically inactivates Yap in the germ cells. The inactivation of Yap in spermatogonia was found to be highly efficient in this model. The loss of Yap in the germ cells had no observable effect on spermatogenesis in vivo. Histological examination of the testes showed no structural differences between mutant animals and age-matched Yapflox/flox controls, nor was any differences detected in gonadosomatic index, expression of germ cell markers or sperm counts. Cluster-forming assay using undifferentiated spermatogonia, including spermatogonial stem cells (SSCs), also showed that YAP is dispensable for SSC cluster formation in vitro. However, an increase in the expression of spermatogenesis and oogenesis basic helix-loop-helix 1 (Sohlh1) and neurogenin 3 (Ngn3) was observed in clusters derived from Yapflox/flox ; Ddx4cre/+ animals. Taken together, these results suggest that YAP fine-tunes the expression of genes associated with spermatogonial fate commitment, but that its loss is not sufficient to alter spermatogenesis in vivo.

Erasure of DNA methylation, genomic imprints, and epimutations in a primordial germ-cell model derived from mouse pluripotent stem cells.

The genome-wide depletion of 5-methylcytosines (5meCs) caused by passive dilution through DNA synthesis without daughter strand methylation and active enzymatic processes resulting in replacement of 5meCs with unmethylated cytosines is a hallmark of primordial germ cells (PGCs). Although recent studies have shown that in vitro differentiation of pluripotent stem cells (PSCs) to PGC-like cells (PGCLCs) mimics the in vivo differentiation of epiblast cells to PGCs, how DNA methylation status of PGCLCs resembles the dynamics of 5meC erasure in embryonic PGCs remains controversial. Here, by differential detection of genome-wide 5meC and 5-hydroxymethylcytosine (5hmeC) distributions by deep sequencing, we show that PGCLCs derived from mouse PSCs recapitulated the process of genome-wide DNA demethylation in embryonic PGCs, including significant demethylation of imprint control regions (ICRs) associated with increased mRNA expression of the corresponding imprinted genes. Although 5hmeCs were also significantly diminished in PGCLCs, they retained greater amounts of 5hmeCs than intragonadal PGCs. The genomes of both PGCLCs and PGCs selectively retained both 5meCs and 5hmeCs at a small number of repeat sequences such as GSAT_MM, of which the significant retention of bisulfite-resistant cytosines was corroborated by reanalysis of previously published whole-genome bisulfite sequencing data for intragonadal PGCs. PSCs harboring abnormal hypermethylation at ICRs of the Dlk1-Gtl2-Dio3 imprinting cluster diminished these 5meCs upon differentiation to PGCLCs, resulting in transcriptional reactivation of the Gtl2 gene. These observations support the usefulness of PGCLCs in studying the germline epigenetic erasure including imprinted genes, epimutations, and erasure-resistant loci, which may be involved in transgenerational epigenetic inheritance.

Indirect effects of Wnt3a/ß-catenin signalling support mouse spermatogonial stem cells in vitro.

Proper regulation of spermatogonial stem cells (SSCs) is crucial for sustaining steady-state spermatogenesis. Previous work has identified several paracrine factors involved in this regulation, in particular, glial cell line-derived neurotrophic factor and fibroblast growth factor 2, which promote long-term SSC self-renewal. Using a SSC culture system, we have recently reported that Wnt5a promotes SSC self-renewal through a ß-catenin-independent Wnt mechanism whereas the ß-catenin-dependent Wnt pathway is not active in SSCs. In contrast, another study has reported that Wnt3a promotes SSC self-renewal through the ß-catenin-dependent pathway, as it can stimulate the proliferation of a spermatogonia cell line. To reconcile these two contradictory reports, we assessed Wnt3a effects on SSCs and progenitor cells, rather than a cell line, in vitro. We observed that Wnt3a induced ß-catenin-dependent signalling in a large subset of germ cells and increased SSC numbers. However, further investigation revealed that cell populations with greater ß-catenin-signalling activity contained fewer SSCs. The increased maintenance of SSCs by Wnt3a coincided with more active cell cycling and the formation of germ cell aggregates, or communities, under feeder-free conditions. Therefore, the results of this study suggest that Wnt3a selectively stimulates proliferation of progenitors that are committed to differentiation or are in the process of exiting the SSC state, leading to enhanced formation of germ cell communities, which indirectly support SSCs and act as an in vitro niche.

CTNNB1 signaling in sertoli cells downregulates spermatogonial stem cell activity via WNT4.

Constitutive activation of the WNT signaling effector CTNNB1 (ß-catenin) in the Sertoli cells of the Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) mouse model results in progressive germ cell loss and sterility. In this study, we sought to determine if this phenotype could be due to a loss of spermatogonial stem cell (SSC) activity. Reciprocal SSC transplants between Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) and wild-type mice showed that SSC activity is lost in Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testes over time, whereas the mutant testes could not support colonization by wild-type SSCs. Microarray analyses performed on cultured Sertoli cells showed that CTNNB1 induces the expression of genes associated with the female sex determination pathway, which was also found to occur in Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) testes. One CTNNB1 target gene encoded the secreted signaling molecule WNT4. We therefore tested the effects of WNT4 on SSC-enriched germ cell cultures, and found that WNT4 induced cell death and reduced SSC activity without affecting cell cycle. Conversely, conditional inactivation of Wnt4 in the Ctnnb1(tm1Mmt/+);Amhr2(tm3(cre)Bhr/+) model rescued spermatogenesis and male fertility, indicating that WNT4 is the major effector downstream of CTNNB1 responsible for germ cell loss. Furthermore, WNT4 was found to signal via the CTNNB1 pathway in Sertoli cells, suggesting a self-reinforcing positive feedback loop. Collectively, these data indicate for the first time that ectopic activation of a signaling cascade in the stem cell niche depletes SSC activity through a paracrine factor. These findings may provide insight into the pathogenesis of male infertility, as well as embryonic gonadal development.

Wnt5a is a cell-extrinsic factor that supports self-renewal of mouse spermatogonial stem cells.

The maintenance of spermatogonial stem cells (SSCs) provides the foundation for life-long spermatogenesis. Although glial-cell-line-derived neurotrophic factor and fibroblast growth factor 2 are crucial for self-renewal of SSCs, recent studies have suggested that other growth factors have important roles in controlling SSC fate. Because ß-catenin-dependent Wnt signaling promotes self-renewal of various stem cell types, we hypothesized that this pathway contributes to SSC maintenance. Using transgenic reporter mice for ß-catenin-dependent signaling, we found that this signaling was not active in SSCs in vitro and in most spermatogonia in vivo. Nonetheless, a pan-Wnt antagonist significantly reduced SSC activity in vitro, suggesting that some Wnt molecules exist in our serum-free culture system and contribute to SSC maintenance. Here, we report that Wnt5a promotes SSC activity. We found that Wnt5a-expressing fibroblasts supported SSC activity better than those not expressing Wnt5a in culture, and that recombinant Wnt5a stimulated SSC maintenance. Furthermore, Wnt5a promoted SSC survival in the absence of feeder cells, and this effect was abolished by inhibiting the Jun N-terminal kinase cascade. In addition, Wnt5a blocked ß-catenin-dependent signaling. We detected the expression of Wnt5a and potential Wnt5a receptors in Sertoli cells and stem/progenitor spermatogonia, respectively. These results indicate that Wnt5a is a cell-extrinsic factor that supports SSC self-renewal through ß-catenin-independent mechanisms.

Soluble growth factors stimulate spermatogonial stem cell divisions that maintain a stem cell pool and produce progenitors in vitro.

Spermatogonial stem cells (SSCs) support life-long spermatogenesis by self-renewing and producing spermatogonia committed to differentiation. In vitro, SSCs form three-dimensional spermatogonial aggregates (clusters) when cultured with glial cell line-derived neurotrophic factor (GDNF) and fibroblast growth factor 2 (FGF2); serial passaging of clusters results in long-term SSC maintenance and expansion. However, the role of these growth factors in controlling patterns of SSC division and fate decision has not been understood thoroughly. We report here that in a short-term culture, GDNF and FGF2 increase the number of dividing SSCs, but not the total SSC number, compared to a no-growth-factor condition. Since the total germ cell number increases with growth factors, these results suggest that GDNF and FGF2 promote a SSC division pattern that sustains the size of the stem cell pool while generating committed progenitors. Our data also show that SSC numbers increase when the cluster structure is disintegrated and cell-cell interaction in clusters is disrupted. Collectively, these results suggest that in this culture system, GDNF and FGF2 stimulate SSC divisions that promote self-renewal and differentiation in the SSC population, and imply that the destruction of the cluster structure, a potential in vitro niche, may contribute to SSC expansion.

Effects of chemotherapeutic agents for testicular cancer on rat spermatogonial stem/progenitor cells.

Spermatogonial stem cells (SSCs) are responsible for the production of spermatozoa throughout adulthood and for the recovery of spermatogenesis following exposure to cytotoxic agents. Previously, we have shown that the combined administration of bleomycin, etoposide, and cisplatin (BEP) used in the treatment of testicular cancer causes impaired spermatogenesis and reduced sperm production in the rat. However, definitive evidence about the potential impact of such chemotherapy on SSCs is still lacking. The objective of this study was to determine whether chronic exposure to BEP treatment causes adverse effects on rat SSC activity. We first investigated the effects of BEP treatment on the clonal organization of undifferentiated spermatogonia by staining whole-mount preparations of rat seminiferous tubules for GFRA1 and ZBTB16 (previously known as PLZF), 2 established markers of undifferentiated spermatogonia. We found that BEP treatment drastically reduced the number of A-aligned spermatogonia while sparing A-single and A-paired cells from the effect. Next, we determined the SSC activity following BEP exposure. Adult transgenic rats carrying EGFP expression in the germ line were treated with BEP for 9 weeks, and SSCs were quantified using spermatogonial transplantation. We found that BEP treatment significantly decreased SSC numbers, which were restored to the control level after a 9-week recovery period. These results demonstrate that BEP treatment transiently affects the activity of rat SSCs.

Development of a short-term fluorescence-based assay to assess the toxicity of anticancer drugs on rat stem/progenitor spermatogonia in vitro.

In vitro culture of rodent spermatogonial stem cells (SSCs) has become an important asset in the study of mammalian SSC biology. Supported by added growth factors, SSCs divide in culture and form aggregates of stem/progenitor spermatogonia, termed clusters. Recent studies have shown that serial passaging of clusters results in long-term maintenance and amplification of the SSC pool and that this culture system can also be used for short-term semiquantification of SSC activity. Here, we report the development of an automated assay to assess the activity of rat stem/progenitor spermatogonia in vitro and its application for investigating the cytotoxicity of chemotherapeutic drugs on these cells. Cultures of EGFP-expressing rat spermatogenic cells allowed us to determine the number and two-dimensional surface area of clusters using an automated fluorescence imaging system, thereby providing quantitative data of SSC activity. Using this assay, we examined the germ cell toxicity of three drugs that are routinely used in testicular cancer therapy, namely, bleomycin, cisplatin, and etoposide, alone and in combination. All three drugs showed a significant and dose-dependent reduction of cluster number and surface area, indicating their adverse effects specific to spermatogonia. The inhibitory concentration at which cluster number and surface area are inhibited by 50% (IC(50)) was the lowest with etoposide and the highest with cisplatin, implying that etoposide was most toxic to spermatogonia in vitro. These results suggest that the SSC culture should provide an effective and efficient system to assess the germ cell toxicity of various drugs and chemical compounds.

The application of biomarkers of spermatogonial stem cells for restoring male fertility.

Spermatogonial stem cells (SSCs) are defined by their ability to both self-renew and produce differentiated germ cells that will develop into functional spermatozoa. Because of this ability, SSCs can reestablish spermatogenesis after testicular damage caused by cytotoxic agents or after transplantation into an infertile recipient. Therefore, SSCs are an important target cell for restoring male fertility, particularly for cancer patients who have to undergo sterilizing cancer therapies. In the mouse, the identification of SSC markers allows for the isolation of a highly enriched population of stem cells. This enriched stem cell population can be expanded in culture for an indefinite period of time, cryopreserved, and transplanted into infertile recipients to restore fertility. Thus, the identification of markers and the establishment of a long-term culture system for human SSCs will be crucial for realizing the potential of these cells in a clinical setting. In this article, we focus on the markers that have been identified for mouse SSCs and discuss how human SSC markers may be used in the restoration of fertility.

Establishment of a short-term in vitro assay for mouse spermatogonial stem cells.

Spermatogonial stem cells (SSCs) are responsible for life-long, daily production of male gametes and for the transmission of genetic information to the next generation. Unequivocal detection of SSCs has relied on spermatogonial transplantation, in which functional SSCs are analyzed qualitatively and quantitatively based on their regenerative capacity. However, this technique has some significant limitations. For example, it is a time-consuming procedure, as data acquisition requires at least 8 weeks after transplantation. It is also laborious, requiring microinjection of target cells into the seminiferous tubules of individual testes. Donor-recipient immunocompatibility for successful transplantation and large variations in data obtained represent further limitations of this technique. In the present study, we provide evidence that a recently developed SSC culture system can be employed as a reliable, short-term in vitro assay for SSCs. In this system, donor cells generate three-dimensional structures of aggregated germ cells (clusters) in vitro within 6 days. We show that each cluster originates from a single cell. Thus, by counting the clusters, cluster-forming cells can be quantified. We observed a strong linear correlation between the numbers of clusters and SSCs over extended culture periods. Therefore, cluster numbers faithfully reflect SSC numbers. These results indicate that by simply counting the number of clusters, functional SSCs can be readily detected within 1 week in a semi-quantitative manner. The faithfulness of this in vitro assay to the transplantation assay was further confirmed under two experimental situations. This in vitro cluster formation assay provides a reliable short-term technique to detect SSCs.

Male germ line stem cells have an altered potential to proliferate and differentiate during postnatal development in mice.

Spermatogonial stem cells (SSCs) continuously support spermatogenesis after puberty. However, accumulating evidence suggests that SSCs differ functionally during postnatal development. For example, mutant mice exist in which SSCs support spermatogenesis in the first wave after birth but cease to do so thereafter, resulting in infertility in adults. Studies using a retroviral vector have shown that the vector transduces pup SSCs more efficiently than adult SSCs, which suggests that pup SSCs divide more frequently. Thus, it is hypothesized that the SSCs in pup and adult testes have different characteristics. As an approach to testing this hypothesis in the present study, we investigated the proliferation kinetics of pup SSCs (6-9 days old) and their self-renewal/differentiation patterns for the first 2 mo after transplantation, and compared them to those of adult SSCs. Using serial transplantation, we found that the number of pup SSCs declined over the first week after transplantation. Thereafter, it increased ~4-fold by 1 mo and ~9-fold by 2 mo after transplantation, which indicates that pup SSCs continuously proliferate from 1 wk to 2 mo after transplantation. Compared to the proliferation of SSCs derived from adult intact testes, that of pup SSCs was lower at 1 mo but similar at 2 mo, indicating the delayed proliferation of pup SSCs. However, the pup SSCs regenerated spermatogenic colonies at 1 mo that were similar in length to those of SSCs from adult intact testes. Therefore, these results suggest that some functional differences exist in SSCs during postnatal development, and that these differences may affect the abilities of SSCs to self-renew and differentiate.

Aging of male germ line stem cells in mice.

In the present study, we investigated the effect of aging on spermatogonial stem cells (SSCs) and on the testicular somatic environment in ROSA26 mice. First, we examined testis weights at 2 mo, 6 mo, 1 yr, and 2 yr of age. At 1 and 2 yr, bilateral atrophied testes were observed in 50% and 75% of the mice, respectively; the rest of the mice had testis weights similar to those of young mice. Next, we evaluated the number and the activity of aged SSCs using spermatogonial transplantation. Numbers of SSCs in atrophied testes decreased in an age-dependent manner to as low as 1/60 of those in testes of young mice. Numbers of SSCs in nonregressed testes were similar regardless of age. The colony length, which is indicative of the potential of SSCs to regenerate spermatogenesis, was similar with donor cells from atrophied testes of 1-yr-old mice and those from testes of young mice, suggesting that SSCs remaining in 1-yr atrophied testes were functionally intact. Colonies arising from SSCs derived from 2-yr atrophied testes were significantly shorter, however, indicating that both SSC numbers and activity declined with age. Finally, we transplanted donor cells from young animals into 1- and 2-yr atrophied testes. Although the weight of 2-yr testes did not change after transplantation, that of 1-yr testes increased significantly, indicating that 1-yr, but not 2-yr, atrophied testes are permissive for regeneration of spermatogenesis by SSCs from young mouse testes. These results demonstrate that both SSCs and somatic environment in the testis are involved in the aging process.

Expression patterns of cell-surface molecules on male germ line stem cells during postnatal mouse development.

Spermatogonial stem cells (SSCs) are stem cells of the male germ line. In mice, SSCs are quiescent at birth but actively proliferate during the first postnatal week, while they rarely divide in adult, suggesting an age-dependent difference in SSC characteristics. As an approach to evaluate this possibility, we studied the expression pattern of cell-surface molecules on neonatal, pup, and adult mouse SSCs. Using immunomagnetic cell sorting, testis cells were selected for the expression of alpha(6) integrin, alpha(v) integrin, c-kit receptor tyrosine kinase (Kit), or a binding subunit of glial-cell-line-derived neurotrophic factor (GDNF) receptor, GFRalpha1. Selected cells were assayed for their stem cell activity using spermatogonial transplantation. The results showed that SSCs expressed alpha(6) integrin, but not alpha(v) integrin and Kit, regardless of age. The SSC activity in pup GFRalpha1(+) cells was higher than that in adult and neonatal cells, indicating that the expression pattern of GFRalpha1 varied age-dependently. To evaluate if SSCs show an age-dependent difference in their response to GDNF, we cultured highly enriched pup and adult SSCs with GDNF: we could not observe such an age-dependent difference in vitro. In addition, we failed to immunologically detect the expression of two types of GDNF receptor signaling subunits on SSCs. These results indicate that SSCs may change the expression patterns of cell-surface molecules during postnatal development, and suggest that GDNF receptor molecules may not be abundantly or specifically expressed in the in vivo population of mouse SSCs.

Genetic analysis of the clonal origin of regenerating mouse spermatogenesis following transplantation.

Spermatogonial transplantation provides a straightforward approach to quantify spermatogonial stem cells (SSCs). Because donor-derived spermatogenesis is regenerated in the form of distinct colonies, the number of functional SSCs can be obtained by simply counting the number of colonies established in recipient testes. However, this approach is legitimate only when one colony arises from one stem cell (one colony-one stem cell hypothesis). In this study, we evaluated the validity of this hypothesis. Two populations of donor cells were obtained from the testes of two transgenic mouse lines and mixed at a 1:1 ratio. Following transplantation of the cell mixture, donor-derived colonies were visualized and individually excised, and genomic DNA was extracted from each colony. Based on unique marker genes of the two transgenic lines, the genotype of the cells contained in a colony was examined by polymerase chain reaction. A colony was determined to be clonal when only one transgene was detected. The results showed that 100% and 90% of colonies were clonal when <5 and 19 colonies were formed per recipient testis, respectively. However, the clonality of colonies decreased as the colony number per recipient testis or the length of each colony increased. These results support the one colony-one stem cell hypothesis and demonstrate that spermatogonial transplantation provides a highly quantitative assay for SSCs; however, these conclusions are applicable under a defined transplantation condition.